This proposal is concerned with how stochastic, initial differences arise in the activity of Notch, which must be properly regulated in development and which, when aberrantly regulated, causes cancer. Stochastic events are utilized from bacteria to humans to mediate myriad developmental processes including cell-fate switching and the generation of cell type diversity. Understanding how stochastic events influence cell fate specification is important to understand how reproducible cell fates are generated from groups of equivalent cells during development. The paradigmatic anchor cell (AC)/ventral uterine (VU) cell fate decision in Caenorhabditis elegans incorporates a stochastic mechanism as part of the process by which initially equivalent cells acquire different fates. The two cells undergoing the AC/VU decision each have the potential to become either an AC or a VU. They interact with each other, mediated by LIN-12/Notch, such that only one becomes the AC and one becomes the VU. During the course of the decision, a stochastic event causes these cells, which initially express both the transmembrane receptor LIN-12/Notch and the transmembrane ligand LAG-2 (a Delta/Serrate/LAG-2 family member), to engage feedback mechanisms that restrict transcription of lin-12 to the presumptive VU and lag-2 to the presumptive AC. The goal of this research is to understand how small, early differences in Notch activity are generated between initially equivalent cells. Birth-order of the AC and VU precursors, a random event, is highly correlated with the subsequent cell fate, suggesting that there is a relationship between cell-cycle progression, Notch signaling, and cell fate specification in the AC/VU decision. Here, we will study this relationship to probe the nature of the stochastic cellular events leading to the specification of the AC and VU by combining the powerful genetics of C. elegans with recent advances in C. elegans genome engineering and in vivo fluorescent imaging. First, we will test whether birth-order of the AC and VU precursors creates or reflects a difference between them by genetically manipulating birth-order and lin-12/Notch level. Second, we will quantify lin-12/Notch expression and activation during the AC/VU decision. We will quantify endogenous lin-12/Notch mRNA and protein expression and develop a novel reporter for lin-12/Notch activity. Third, we will conduct genetic screens to identify novel regulators that control Notch activation i the AC/VU decision and their sister cells by directly imaging the somatic gonad at high magnification. In addition to addressing a central question in developmental biology, studies of Notch-mediated cell fate decisions are highly relevant to cancer: Notch can be an oncogene or tumor suppressor depending on cell context, and studies of how Notch activity is regulated in this cell fate paradigm in C. elegans have identified genes involved in tumorigenesis. Furthermore, illuminating the potential intersection between Notch activity, cell cycle progression, and cell fate specification may suggest novel therapeutic approaches.